Isolation of alpha1 -fetoprotein

ABSTRACT

A method of isolating alpha 1  -fetoprotein sufficiently pure to be useful in radioimmunoassay procedures is described. The method comprises the procedures and techniques of affinity chromatography, immobilized reactive dye adsorption chromatography and polyacrylamide gel electrophoresis. Wherein a certain degree of reduction in kind and/or quantity of contaminants is not achieved by the first two procedures, gel filtration and mixed ion exchange cellulose chromatography are carried out in preparation for polyacrylamide gel electrophoresis. Monkey alpha 1  -fetoprotein very similar to human cord blood alpha 1  -fetoprotein by amino acid composition and physical properties and of sufficient purity to be useful in human radioimmunoassay procedures is obtained by the disclosed method.

RELATED APPLICATIONS

This application is a continuation-in-part of U.S. patent applicationSer. No. 846,089, filed Oct. 27, 1977, abandoned.

BACKGROUND OF THE INVENTION AND STATEMENT OF PRIOR ART

A serum protein specific to the fetus was first demonstrated byPederson, Nature (London) 154, 575 (1944) in calf serum and by Bergstandet al. Scand. J. Clin. Lab. Invest 8 174 (1956) in humans. Thisfeto-specific protein was designated alpha₁ -fetoprotein (AFP) due toits electrophoretic mobility. Alpha₁ -fetoprotein in sera ofhepatocellular cancer and malignant embryonal teratoma patients wasfound to be immunologically and electrophoretically identical withalpha₁ -fetoprotein observed in fetal serum by Tatarinov, Vopr. Med.Khim. 11 20-24 (1965) and Abelev et al., Int. J. Cancer 2 551-558(1967).

Human alpha₁ -fetoprotein has been isolated from fetal serum by chemicalprocedures by Pederson, Clin. Chimica Acta 38 163-170 (1971), Ruoslahitet al., Int. J. Cancer 7 218-225 (1971), Silver et al., Proc. Nat. Acad.Sci., U.S.A. 70 526-530 (1973), Forrester et al., Clin. Chimica Acta 64317-323 (1975) and Twomey et al., Clin. Chem. 22 1306-1309 (1976). Inaddition immuno-chemical and chemical methods have been employed toisolate alpha₁ -fetoprotein from hepatoma serum by Nishi, Cancer Res. 302507-2513 (1970), Nishi et al., Protides Biol. Fluids 18 43-47 (1971),Nishi et al., Biochem. Biophys. Acta 278 293-298 (1972), Aoyagi et al.,Cancer Research 37 3663-3667 (Oct. 1977), Yachnin et al., Biochem.Biophys. Acta 493 418-428 (1977), Lehmann et al., Clin. Chimica Acta 33197-206 (1971) and Alpert et al., J. Biol. Chem 247 3492-3497 (1972).

The methods referred to above result in an alpha₁ -fetoprotein (AFP)which is not pure in that it contains albumin and other proteins.Because of the presence of these contaminants, such relatively impurealpha₁ -fetoprotein preparations are not satisfactory as theradiolabeled antigen for a sensitive radioimmunoassay. Further, suchpreparations are not satisfactory for the production of monospecifichigh titer antisera for a sensitive radioimmunoassay (RIA). Becausesamples to be tested for AFP in a radioimmunoassy often contain AFP inlow concentration, it is essential that the RIA procedure be highlysensitive. By highly sensitive is meants that the RIA must be able toaccurately detect AFP at a level of about 20 ng/ml of sample. Suchsensitive RIA procedures are required for screening for birth defects inpregnant women, which has heretofore not been feasible.

Non-specific antisera or impure labeled AFP cannot be utilized in suchsensitive RIA procedures since they would not yield an accurate measureof the AFP content of the sample. In addition, the methods of alpha₁-fetoprotein reported in the literature are not satisfactory forisolating AFP from large volumes of source solutions and/or sourcesolutions which contain low concentrations of AFP. Further, the methodsdisclosed in the literature for isolating AFP do not appear to besatisfactory for automation techniques.

Specifically, the Ruoslahti et al. article teaches a method forpurifying AFP from fetal serum utilizing two electrofocusing proceduresin series or immunoprecipitation. The electrofocusing procedures arebasically separations based on charge. There is no disclosure that theAFP obtained by the disclosed procedure would be of sufficient purity tobe utilized in a RIA.

Alpert et al. teach a method of purifying AFP by a three step chemicalprocess including: (1) starch gel block electrophoresis, (2) gelfiltration utilizing a Sephadex column, and (3) isoelectric focusing.The AFP thus obtained is stated to be pure by immunodiffusion and sodiumdodecyl sulfate gel electrophoresis. In addition, the AFP thus obtainedis stated as being capable of producing immunospecific antiserum inrabbits. In contrast to the method of the captioned application whichutilizes immunological as well as chemical techniques, there is nosuggestion that the AFP produced by the disclosed method is sufficientlypure for RIA procedures.

The first two Nishi articles listed above utilize hepatoma serum assource material and purify basically by immunoprecipitation followed bygel filtration.

The method of the third Nishi article also utilizes hepatoma serum as asource material and teaches a purification method utilizing animmunosorbent column. However, the antibody is not adsorbed to insuremonospecificity. Further purification is carried out by gel filtration.

None of the methods disclosed in the Nishi articles appears to teach oneskilled in the art how to purify AFP to a degree sufficient for use inRIA procedures. Certainly, none contains even the slightest suggestionthat AFP can be obtained from monkey source material in sufficientpurity for use in human RIA procedures.

Aoyagi et al. teach a method of purifying AFP from cord serum utilizingan immunosorbent column, ion exchange on DEAE Sephadex and gelfiltration. Again, there is no indication of the AFP purified by thisprocedure being suitable for RIA procedures. Although ion exchange andgel filtration chromatography does not afford optimum separation ofalbumin due to closeness of charge and molecular weight, the AFPproduced by the disclosed procedure appears to be relatively pure. Thereis no suggestion, however, that the disclosed method could produce AFPsuitable for human RIAs from monkey source material in contrast to themethod of the present invention.

Yachnin et al. disclose a method of purifying AFP hapatoma serumutilizing an immunosorbent column and gel filtration. It appears fromthe disclosure that the product is microheterogeneous and, therefore,unsatisfactory for RIA procedures.

There is thus a need for a method of isolating alpha₁ -fetoprotein in aspure a state as possible in terms of albumin and other proteincontaminants which would interfere with sensitive radioimmunoassays.Further, there is a need for isolating AFP which can be efficientlyapplied to large volumes of source solutions and/or source solutionswhich contain AFP in low concentration. Finally, there is a need for amethod of isolating AFP which is amenable to being carried out, whollyor partially, by automated apparatus and techniques. These needs are allsatisfied by the method of the subject invention. Further, themethodology of the present invention affords a means whereby AFP can beobtained from monkey hepatoma serum in sufficient purity so as to be forpractical purposes, the same as that obtained from human cord serum andsufficiently pure to be used in RIA procedures on human samples. MonkeyAFP has heretofore not been recognized as being usable inradioimmunoassays on human material.

DESCRIPTION OF THE INVENTION

This invention provides a method for isolating alpha₁ -fetoprotein frombiological source material such as cord blood, hepatoma tissue andextracts thereof, other tumors, ascities fluid, fetal blood, embryonictissues, amniotic fluid hepatoma blood, and cells grown in culture or inhost animals. Further, the present pertains to a method of purifyingalpha₁ -fetoprotein (AFP) from monkey sources, particularly monkeyhepatoma blood. It has been found that AFP purified from this sourceaccording to the invention demonstrates immunologic identity with AFPpresent in human cord blood and human ascities fluid. The use of monkeysource material such as hepatoma blood for the isolation of pure AFP bythe method of the invention is significant because such material is moreuniform and significantly higher in AFP content than certain othersources such as human cord blood. AFP concentration in monkey hepatomablood ranges from about 1 to about 5 mg./ml. as opposed to human cordblood which contains from about 0.06 to about 0.1 mg./ml.

The process of this invention provides a pure alpha₁ -fetoprotein (AFP)essentially free of albumin and other protein contaminants and which canbe used in a sensitive radioimmunoassay. The process involves using thefollowing techniques in sequence:

(a) affinity chromatography on an immunosorbent column;

(b) immobilized reactive dye adsorption chromatography;

(c) gel filtration;

(d) mixed ion exchange cellulose chromatography and

(e) polyacrylamide gel electrophoresis.

The resulting product, alpha₁ -fetoprotein, is homogenous, i.e., appearsas a single band, by analytical disc electrophoresis. Uponimmunoelectrophoresis, AFP obtained by the method of the subjectinvention demonstrates an electrophoretic mobility in the alpha₁-globulin region, demonstrates a single precipitin line against hightitered anti-normal human serum or anti-human serum albumin. Theseobservations have been confirmed by radioimmunoelectrophoresis. AFPpurified in accordance with the subject invention demonstrates a line ofidentity in double immunodiffusion with alpha₁ -fetoprotein in cordserum, hepatocellular cancer serum and ascities fluid and is essentiallyfree of albumin an other proteins which would interfere with its use insensitive radioimmunoassays (RIAs).

The novel process of this invention involves a combination ofimmunological, chemical, filtration, electrostatic and electrophoreticmethods and techniques.

Affinity Chromatography

In order to remove the bulk of the extraneous proteins from an alpha₁-fetoprotein containing sample of source solution such as, for example,human cord serum or monkey hepatoma serum, the sample is initiallysubjected to affinity chromatography.

Affinity chromatography is an immunological adsorption technique whereinthe antigen is removed from solution by being adsorbed onto animmunosorbent which comprises antibodies specific to the antigen to beisolated covalently coupled to a solid, particulate support. Proteinswhich do not react with the immobilized antibody are not bound to thesolid support. These unbound contaminants are therefore easily separatedfrom the bound antigen. The antigen is thereafter separated from theantibody which remains bound to the solid support.

In order to assure that the affinity chromatography is specific to theantigen sought, the immunosorbent must be treated to block thenon-specific sites remaining after the antibody is coupled thereon. Inorder to accomplish this, the immunosorbent formed by coupling theantibody to a solid support is first equilibrated with a suitablebuffer. The immunosorbent is then treated sequentially with normal humanserum, again with the same buffer utilized to equilibrate and withammonium thiocyanate. The immunosorbent is then reequilibrated utilizingthe same buffer.

After the the immunosorbent is equilibrated, the sample is passedthrough the column and the antigen, AFP, is adsorbed unto its antibodywhile extraneous proteins are eluted therefrom. The sample is initiallydiluted with buffer at pH 7.0 so that a theoretical AFP concentration offrom about 0.05 to about 0.1 mg./ml. is obtained. In the case of, forexample, cord serum a 1:1 volume dilution is formed with the buffer.After the sample has been passed through the column, the antigen iseluted by use of a desorbing agent which breaks the antigen-antibodycomplex. Suitable desorbing agents include, for example, 6 to 8 molarsolutions of urea, aqueous sodium chloride solutions having a highconcentration, i.e., 0.5 molar and above, acid buffers, i.e., buffershaving a pH of from about 2 to about 3 and buffers containing chaotropicions, i.e., ions known to possess the ability to break antigen-antibodybonds such as, for example, the thiocyanate ion. A preferred desorbingagent is 3.0 M ammonium thiocyanate in 0.1 M sodium phosphate buffer atpH 7.0. The desorption of the AFP from the chromatography columnregenerates the column which requires only equilibration with a suitablebuffer for reuse.

The combined eluates of the adsorbed fraction, i.e., the AFP-containingfraction, are then dialyzed against deionized water and the resultingdialysate concentrated by utrafiltration. The adsorbed fraction is thentested for AFP content by immunologic techniques.

The materials which are suitable for use as solid supports for theantibody must meet a number of critical parameters, i.e., they must

(a) immobilize gamma globulin (antibodies) without adversely affectingtheir immunological properties,

(b) permit a sample of test fluid to pass through a column at anacceptable rate,

(c) be inert, i.e., they must have no immunologically interferingproperties or be capable of being modified chemically or immunologicallyso as to have no such interfering properties,

(d) permit desorption of antigens without the antibody being desorbed oraffected immunologically; and

(e) be suitable for use in an automated system.

Materials which have these properties include, for example, agarosegels, porous glass beads, polyacrylamide beads and the like, preferablyagarose gels. Agarose is the neutral portion of agar. Agarose gel iscommercially available, for example, under the trademark "Sepharose"from AB Pharmacia, Uppsala, Sweden. The commercially available agarosegels are aqueous suspensions usually containing a preservative such as,for example, 0.02% sodium azide. The gel is prepared in bead form havinga selected particle size and percent concentration of agarose. Theconcentration of the agarose in the gel determines its fractionationrange.

The gels most suitable for use in the affinity chromatography step ofthis i.e., which meet all of the above-mentioned criteria, are availableunder the trademark "Sepharose 4B". These gels have a particle size offrom 40-190 microns and contain 4% by weight agarose. Further, thesegels have a fractionation range of 3×10⁵ to 3×10⁶.

The buffer preferred for use in the affinity chromatography step of thisinvention is a 0.1 M phosphate buffer at pH 7.0. Other buffers whichmight be utilized are, for example, borate buffers at pH 8, acetatebuffers at pH of about 5 and the like. The choice of a particular bufferis governed by factors as, for example, the stability of the antigenbeing isolated and the like.

The preferred method of concentrating the dialysate by ultrafiltrationis to use a stirred cell containing a membrane having a molecular weightcut off of 10,000, such as a PM 10 membrane available from Amicon Corp.,Lexington, Mass. It is also possible to concentrate the dialysate byother methods known in the art such as, for example, freeze drying.

The AFP content in the resulting material can be monitored by severalimmunological assay means, however, radioimmunoassay (RIA) is preferred.Where feasible, the addition of a small measured amount of radiolabelledAFP to the partially purified sample and the monitoring of its passagethrough the various stages of the process facilitates and insures thepurity of the final preparation.

The affinity chromatography step of the process of this invention can beautomated by using a modified version of a device named "CYCLUM" whichwas developed at Oak Ridge National Laboratory, Oak Ridge, Tenn. TheCYCLUM control panel with its associated equipment is programmed by theuse of a cam timer to select one of three liquids to flow through anaffinity column, monitor and record the optical density of the effluentand dispense the effluent into one of the three containers. The CYCLUMwhich has the capacity to continuously recycle the affinitychromatography treatment, is described in Anderson et al., CancerResearch, Vol. 34, pp. 2066 to 2076 (1974).

Immobilized Reactive Dye Adsorption Chromatography

Albumin present in the adsorbed fraction produced in the previous stepis substantially removed from the AFP by means of a immobilized reactivedye adsorbent. Examples of suitable dyes are the so-called reactive dyesof, for example, the Cibacron series. These dyes are derived fromcyanuric chloride, i.e., 2, 4, 6-trichloro-1, 3, 5-triazine. The dyessuited for the practice of the present invention are sulfonated,poly-aromatic dyes containing a 1, 3, 5-triazine nucleus and a singlereactive chlorine. Examples of suitable dyes includes Cibacron BrilliantBlue, Cibacron Scarlet and Cibacron Brilliant Orange. Preferred isCibacron Brilliant Blue which has the structure ##STR1##

The reactive dye is immobilized by being bound, i.e., covalently linkedor adsorbed onto a solid support such as those discussed above inreference to affinity chromatography. A preferred immobilized reactivedye in accordance with the present invention is Sepharose-blue dextran.Sepharose-blue dextran is prepared by co-valently linking blue dextran,i.e., dextran having Cibacron Brilliant Blue chemically bound thereto toagarose gel, e.g., Sepharose 4B. The linking of the blue dextran toSepharose is accomplished by the method of Nishikawa et al. Anal.Biochemistry, Vol. 64, pp. 268-275 (1975). This method involvesactivating the Sepharose with cyanogen bromide buffered at pH 11 atabout 4° C. The blue dextran is then added in 0.1 M phosphate buffer atpH 7 and the reaction allowed to proceed for 24 hours at about 4° C. Theactivated Sepharose is attached to the dye via one of the aromaticgroups therein. The resulting preparation is capable of binding 10 mg.of albumin per ml. of packed gel.

The immobilized reactive dye adsorption chromatography is carried oututilizing a column 5 cm×13 cm. The procedure described in Travis et al.,Clin. Chimica Acta 49 49-52 (1973) may, for example, be used toselectively remove the albumin from the AFP containing fractions.Accordingly, the pooled AFP containing fractions from the affinitycolumn are dialyzed against the column buffer, i.e., 0.05 M TRIS-0.5 MNaCl, pH 8.0, prior to being pumped through the bed of immobilizedreactive dye adsorbent. The unadsorbed eluate which contains AFP, ispooled and concentrated by ultrafiltration or other suitable means as inthe previous step. The resulting AFP containing material is essentiallyfree of albumin as determined by immunologic techniques. About 90% to95% of the albumin originally present in the sample is removed by thistechnique. The immobilized reactive dye adsorption chromatography columnmay be regenerated by treatment with a 6.0 molar urea solution to removethe albumin, followed by equilibration with a suitable buffer.

Gel Filtration Chromatography

The unadsorbed fraction from the immobilized reactive dye adsorptionchromatography column containing AFP is then subjected to gel filtrationchromatography to separate the high molecular weight contaminants and aportion of any remaining albumin. The molecular weights of AFP andalbumin are 72,000 and 69,000 daltons, respectively. By high molecularweight contaminants is meant proteins having a molecular weight of about100,000 or more. The preferred column for this treatment contains afiltering material which is a hydrophilic, water-insoluble, cross-linkeddextran polymer gel. This material and the method of its manufacture aredescribed in British Pat. No. 854,715. The preferred gel material, whichis commercially available from AB Pharmacia, Uppsala, Sweden under thename "Sephadex", comprises a three dimensional macroscopic network ofdextran substances bonded or cross-linked together which is capable ofadsorbing water with swelling. The ability of the gel material to takeup water is inversely proportional to the degree of cross-linkage ofdextran substances in the gel material. The gel material is available ina variety of different grades differing with respect to degree ofporosity. The gel preferred for use in this invention has an approximatemolecular weight exclusion limit of 200,000, a water regain (g. H₂ O/g.dry gel) of 20±2.0, a particle size of 40-120 microns and a bedvolume/ml./gm. dry gel of 30-40. This particular gel is designated"Sephadex G-200".

The gel column is equilibrated with 0.05 M monobasic sodiumphospate--0.15 M sodium chloride buffer, pH 5.0. The unadsorbed fractionfrom the immobilized reactive dye adsorption chromatography column,which contains AFP, is concentrated and dialyzed by discontinuousdiafiltration against the same buffer and then chromatographed on theequilibrated gel column, using upward flow. Upward flow is used becauseit facilitates separation of the AFP from low- and high-molecular weightprotein contaminants and maintains the integrity of the gel bed.

The eluate from the column is collected in fractions of, e.g., 10 ml.These fractions are monitored for protein content and radioactivity. Inaddition, the AFP content of certain fractions is confirmed by RIAprocedure. Generally, about 150 such fractions are obtained. Thefractions having the highest AFP content are collected and pooled. Therest are discarded. Generally, when 150 fractions are collected,fractions 100 to 120 have been found to contain the maximum AFP.

Ion Exchange Cellulose Column Chromatography

In order to further remove contaminating proteins from theAFP-containing fractions from the gel filtration chromatography step,the pooled fractions are subjected to ion exchange cellulose columnchromatography. This procedure separates the proteins by virtue of theirelectrostatic binding capacity as opposed to, e.g, the previouslycarried out gel filtration chromatography which separated the proteinsby size.

The ion exchange column found suitable for use in this procedure is amixed bed column composed of a cationic exchanger, e.g., carboxymethylcellulose and an anion exchanger, e.g., diethylaminoethyl cellulose.

The carboxymethyl celluloses suitable for use in this procedure are, forexample, those which are microgranular in form, have rod shapedparticles with a particle size distribution expressed as the diameter ofequivalent spheres within a range of about 20μto about 60μ, have acapacity of 1.0± meq./gm. and a water regain of 2.3-2.7 gm./gm. dryexchanger. The preferred ionic form is the Na⁺ form. A suitable ionexchanger is commercially available in a dry form from H. Reeve AngelInc., Clifton, N.J., under the trade name "CM 52".

The diethylaminoethyl cellulose preparations most suitable for use inthis procedure are those which are microgranular in form, have rodshaped particles with a particle size distribution expressed as thediameter of equivalent spheres within a range of about 20μ to about 60μ,have a capacity of 1.0±0.1 meq./gm. have a water regain of 2.3-2.8gm./gm., dry exchanger and are in the free base form. A suitable ionexchanger, for example, is that commercially available from H. ReeveAngel Inc., Clifton, N.J. under the trade name "DE 52".

The mixed ion exchange cellulose column is prepared as follows. First,the fines are removed from each exchanger, for example, by aspiration ofthe supernatant resulting from adding a 10-fold volume of water,stirring and allowing the solids to settle. Second, each exchanger isseparately equilibrated with a 2 molar sodium chloride solution in 0.1 Mammonium acetate. After aspiration of the supernatant, a buffer solutionof 0.1 M ammonium acetate is added to each of the celluloses and equalvolumes of each of the resulting slurries are combined and packed bygravity into a 2.5 cm×13.0 cm column. The resulting column isequilibrated in terms of the buffer to be used, i.e., 0.1 M ammoniumacetate.

The pooled fractions from the gel filtration procedure are concentratedand equilibrated by dialysis against the 0.1 M ammonium acetate buffer.The AFP containing solution is then applied to the ion exchange columnat the rate of 60 ml./hr.

After the AFP solution has been applied to the column, the column iswashed with additional buffer to remove any material which does not bindthereto. Thereafter, a linear sodium chloride gradient is established bythe very gradual addition of sodium chloride to the buffer. As the saltconcentration increases in the buffer, 3 ml. fractions are collectedand, as in the previous step, monitored for protein content,radioactivity and for AFP by RIA. Again, the AFP containing fractionsare collected and pooled.

Preparative Polyacrylamide Electrophoresis

Purification of the AFP is completed by polyacrylamide electrophoresis.This procedure, which separates AFP from contaminating proteins on thebasis of differences in molecular charge, size and shape, removes anyremaining interfering traces of albumin which might be present. Thisprocedure is carried out preferably utilizing a Buchler Polyslab(Buchler Instruments, Fort Lee, N.J.) which is a verticle 170 mm×3.0 mmslab of a 7% polyacrylamide resolving gel having a 10 mm×3.0 mmconcentrating gel polymerized on top of the resolving gel.

The difference between the concentrating gel and resolving gel describedabove is basically pore size, the former being larger. Variance in poresize is achieved by varying the relative proportions of monomer, i.e.,acrylamide and comonomer (cross-linking agent), i.e.,N,N'-methylenebisacrylamide in the polymerization mixture. For adescription of these gels, their preparation and use thereof inelectrophoretic procedures, see Davis, "Disc Electrophoresis--II, Methodand Application to Human Serum Proteins". Annal. N.Y. Acad. Sci., Vol.121, pp. 404-427 (1964).

The collected, pooled fractions from the previous procedure are dialyzedagainst water. The dialysate is then lyophilized to a powder. The powderis then dissolve in a small quantity of a buffer termed electrophoresisbuffer. This buffer solution contains about 0.6% by weight TRIS, i.e.,2-amino-2-(hydroxymethyl)-1,3-propanediol and about 3% by weight glycinewith a pH of approximately 8.3. Sucrose and a trace of a suitable dyesuch as, for example, bromophenol blue are then added to the sample. Thebromophenol blue is used as an indicator of the progress of theprocedure as it migrates through the gels ahead of the proteins. Thesucrose is present to increase the density of the sample solutionsomewhat. It is preferred to add sufficient sucrose to achieve aconcentration of about 8% by weight. The AFP containing solution isplaced into the Buchler Polyslab and electrophoresis is carried out for24 hours at 40 mA constant current. During electrophoresis, the proteinsin the layer of sample solution which is 4-6 mm. in height will firstform a thin band, i.e., about 1 mm. in height, near the bottom of theconcentrating gel before migrating through the resolving gel layer.

When electrophoresis is complete, the resolving gel is removed andsliced to 0.5 cm thick strips transverse to the path between the cathodeand the anode. The strips are individually eluted with 0.05 M boratebuffer at pH 8.4. The eluates of the strips are monitored for AFPcontent by radioactivity, double immunodiffusion and RIA.

The above-described five step process is required in accordance with thepresent invention to obtain pure AFP from certain sources such as humancord serum. It is within the purview of the present invention, however,to obtain pure AFP from other sources, particularly monkey hepatomaserum utilizing only three of the five described steps. The choicebetween these three steps, i.e., affinity chromatography, immobilizedreactive dye adsorption chromatography and polyacrylamideelectrophoresis, and the above described five step procedure is made inview of the following considerations.

The choice of a three step vs. a five step procedure for obtaining pureAFP in accordance with the present invention is to be made inconsideration of the nature and/or the quantity of contaminants. Thedetermination of whether three or five steps are required is made afterthe immobilized reactive dye chromatography is carried out. A sample ofthe pooled unabsorbed fractions from immobilized reactive dyechromatography is subjected to analytical disc electrophoresis. If thisprocedure demonstrates contaminants of 20% and above, the five stepprocedure is utilized. Even if the level of contaminants is withinacceptable limits, i.e., less than 20%, a five step procedure isnonetheless utilized if the contaminants demonstrate an electrophoreticmobility similar to that of AFP.

In the event that only a three step purification procedure is required,the pooled, unadsorbed fractions from the immobilized reactive dyechromatography are dialyzed and lyophilized. The resulting powder isthen dissolved in the electrophoresis buffer and subjected topolyacrylamide electrophoresis as described above.

The above-described process, regardless of whether three or five stepsare utilized, yields pure AFP as judged by analytical discelectrophoresis. The pure AFP obtained from e.g., cord serum, ascitesfluid and hepatoma serum, demonstrates immunologic identity inimmunodiffusion. AFP obtained from monkey hepatoma serum likewisedemonstrated immunologic identity with the preparations obtained fromhuman sources. The observation of a single precipitin line inimmunodiffusion for these preparations of purified AFP against aheterospecific unadsorbed rabbit anti-AFP serum demonstrated immunologichomogeneity. The purity of AFP obtained in accordance with the method ofthe present invention was also determined by immunoelectrophoresis ofthe purified AFP against anti-AFP serum (one precipitin line) and hightitered anti-normal human serum anti-human serum albumin (no precipitinline).

The purity of the AFP obtained in accordance with the subject inventionwas determined, in addition to the methods previously mentioned, byspecific activity. The specific activity equals the antibodyneutralization (RIA) divided by the protein concentration and isdescribed by Lowry et al., J. Biol. Chem., Vol. 193, pp. 265-275 (1951).Bovine serum albumin (Pentex, Cat. No. 83-301) was used as the proteinreference standard. The specific activity of the purified AFP preparedin accordance with the present invention was found to range between 0.87and 0.95 for cord serum AFP and between 0.94 and 0.99 for hepatocellularcarcinoma serum AFP. In contrast, the specific activity of the AFPcontaining pooled fractions prior to preparative polyacrylamideelectrophoresis ranged from 0.45 to about 0.75 and yielded aradiolabeled product which was not usable in a RIA procedure due tolarge amounts of iodinated high and low molecular weight contaminants.

The purified AFP prepared in accordance with the subject invention issuited for use in a highly sensitive diagnostic radioimmunoassay forhepatocellular cancer and birth anomalies. AFP previously known to theart is not well suited to such a radioimmunoassay procedure because ofthe amount of albumin present. In addition to facilitating thesubstantial removal of albumin which interferes with sensitive RIAprocedures, the method of the present invention is advantageous overother attempts to purify AFP known to the art in that it is effective inisolating AFP from solutions which contain it in low concentration and,further, that it is effective because of automation in dealing withlarge volumes of such solutions. Further, the method of the subjectinvention provides AFP isolated from monkey hepatoma serum of suchpurity that it is very similar to AFP obtained from human sources byamino acid analysis and is usable in RIA procedures in place of AFP fromhuman sources.

For use in radioimmunoassay procedures, AFP particularly monkey AFP isradiolabeled with radioactive atoms which will react with its chemicallyreactive groups and not substantially diminish its antigenicity. ¹²⁵ Ihas been found to be particularly suitable. AFP can be radioiodinated bymethods known in the art, with minor modifications to concentration andvolume. The Chloramine T method of Hunter and Greenwood described in J.Biochem 91, 46 (1964) using iodine 125 is particularly useful.

The reaction is effected, for example, by mixing 50 μg. AFP in boratebuffer with 5 mCi of Na¹²⁵ I (pH 8-11) and 100 μg. fresh Chloramine T(sodium p-toluenesulfochloramine) all in borate buffer. The reactiontakes place in 1 minute at room temperature and is stopped by theaddition of sodium metabisulfite. The labeled product is mixed with 1ml. of a 5% human serum albumin solution and the mixture applied to across-linked dextran gel column, e.g., Sephadex G-100, to separate theproduct from unreacted ¹²⁵ I. The product is then eluted from the columnwith, e.g., 0.1 M TRIS-0.15 M sodium chloride buffer, pH 7 and collectedin tubes (5 ml. fractions) containing 0.5 ml. of a 5% human albuminsolution. The product is diluted to suitable concentration with boratebuffer containing 0.25% human albumin.

The monkey AFP labeled with ¹²⁵ I as described above is particularlysuited to a radioimmunoassay procedure. That one skilled in the artwould not appreciate that this material could be so utilized is evidentwhen it is considered that, prior to the present invention, AFP had notbeen prepared sufficiently pure for RIA procedures and, further, thatthe prevailing feeling among those skilled in the art has been that onlymaterials of human origin could be used as the labeled source materialfor the quantitative assay of human AFP by RIA. AFP purified inaccordance with the present invention can be used in a highly sensitiveradioimmunoassay for AFP, i.e., a sensitivity of from about 0.5 ngm. toabout 18 ngm.

A preferred RIA utilizing AFP purified in accordance with the presentinvention is a solid phase immobilized second antibody procedure. Inthis procedure rabbit anti-AFP is the primary antibody and goatantiserum, i.e., goat anti-rabbit gamma globulin as the secondaryantibody. The standard material for the assay is partially purified cordblood AFP.

In conducting the radioimmunoassay, a standard competitive-inhibitioncurve is created. It is a measure of the complex formation between theadded antigen with specific antibodies. The curve reflects the amount ofAFP per unit of sample. The measurement is in nanograms per dose peraliquot of standard AFP solution which is plotted against the quantityof antibody complexed with radiolabeled pure AFP. The resulting curve isused to determine the amount of AFP in a sample, i.e., serum, plasma oramniotic fluid.

In a preferred method, a measured standard material is added to a seriesof tubes containing ethylenediaminetetraacetic acid (EDTA) buffer, pHabout 6 which contains about 1% by volume normal goat serum. A measuredamount of primary antibody, i.e., rabbit anti-AFP, diluted with asuitable buffer such as, for example, 0.05 M borate buffer, pH 8.4containing 0.25% by weight human albumin.

Following the addition of dilute antibody, a measured amount ofradioiodinated pure monkey AFP, is added to each tube. The amount addedcan, for example, range between 0.6 ng. and 1.0 ng. The resultingsolutions are incubated at ambient temperature for a sufficient time tocomplete the reaction, usually about 5 hours. When the incubation iscompleted, the immobilized secondary antibody, i.e., goat anti-rabbitgamma globulin, is added to each tube and the solution again incubatedfor an additional period of about 5 minutes at ambient temperature. Theimmobilized antibody complexes with the AFP/primary antibody complexthereby facilitating its removal from solution.

Under the conditions described above, free AFP remains in solution. The¹²⁵ I content of the precipitate or the supernatant is then determinedon a suitable counter and the radioactivity then can be plotted againstthe antigen concentration. In this way a curve is established which canbe utilized to determine the amount of AFP in a sample of plasma, serumor amniotic fluid following the procedure described above.

The secondary antibody can be immobilized by a number of meansrecognized in the art such as, for example, by the methods previouslydescribed with reference to immunosorbent. In addition to beads, glassor polycarbonate rods are also suitable for immobilizing the secondantibody. A preferred method of immobilizing the secondary antibody isto adsorb it onto an unsintered fluorocarbon polymer according to themethod described in Fishman, U.S. Pat. No. 3,843,443 issued Oct. 22,1974. A particularly preferred fluorocarbon polymer for immobilizing thesecondary antibody is unsintered polyvinylidene fluoride.

It is to be understood that the present invention insofar as it pertainsto improvements in RIA procedures is not intended to be limited to theprocedure described above. Any radioimmunoassay procedure recognized inthe art for AFP comprising, basically, forming a complex of the AFP inan unknown with an antiserum, adding thereto a probe, i.e., radiolabeledAFP, removing the resulting complex from solution and measuring theamount of radiolabeled material taken up against a standard can beimproved by using as the radiolabeled material purified AFP prepared inaccordance with the present invention.

The accuracy of the RIA is dependent on the purity of the radiolabeledmaterial. Because radioimmunoassays for AFP such as described above arerendered more sensitive utilizing AFP purified in accordance with thepresent invention, they can now be utilized, for example, in a screeningprogram for birth defects in pregnant woman. The use of an RIA procedurein such a program has heretofore not been considered feasible.

The following Examples further illustrate the invention.

EXAMPLE 1

Pooled human cord blood serum was obtained from term pregnancies andstored at -20° C. The serum was thawed, centrifuged at 100,000 r.p.m.for 1 hour at 4° C. and the supernatant fluid diluted with an equalvolume of 0.1 M sodium phosphate buffer containing 0.02% by weightsodium azide, pH 7.0.

Affinity Chromatography

Portions of 15 ml. of the dilute cord serum prepared above were cycledthrough an automated recycling chromatographic system programed todispense three liquids, i.e., sample, buffer and desorbent into anaffinity column and separately collect waste, unadsorbed fraction andadsorbed fraction. The process was repeated continuously until all ofthe diluted cord serum had been chromatographed through the column. Theadsorbed fractions were collected and dialyzed against deionized waterby passage through two Bio-Rad Hollow Fiber Units (Bio-Rad, Cat.#B/HDG-1) connected in series. The dialysate was pooled and concentratedto a volume of about 20 ml. by ultrafiltration.

The column of the automated chromatographic system was packed with animmunosorbent prepared as follows:

Alpha₁ -fetoprotein was isolated from the serum of a monkey with achemically induced hepatoma. The AFP was purified by affinitychromatography and ascending gel filtration column chromatography andequilibrated with 0.05 M monobasic sodium phosphate--0.15 M sodiumchloride buffer at pH 5.0. New Zealand white rabbits were immunized withthe antigen which had been coupled to methylated bovine serum albuminprior to mixing with complete Freund's adjuvant. The production of hightiter anti-AFP serum was monitored by double immunodiffusion andimmunoelectrophoresis.

The rabbit anti-AFP (monkey) serum thus produced was absorbed with 3mg./ml. of powdered human serum albumin for 1 hour at 37° C. followed by18 hours at 4° C. and centrifugation at 100,000 r.p.m. for 1 hour. Theresulting 30 ml. of adsorbed antibody was subjected to descending DEAEcellulose column chromatography on a 2.5 cm×40.0 cm column in 0.01 Msodium phosphate buffer at pH 8.0. The antibody-containing fractionswere pooled and covalently linked to 50 ml. of Sepharose 4B (Pharmacia)activated with cyanogen bromide dissolved in N-methyl-2-pyrrolidone. Thegamma globulin fraction was added to the activated Sepharose 4B and thereaction was allowed to proceed for 24 hours at 4° C. After mixing withan equal volume of aqueous 1 M ethanolamine for one hour at pH 8.0, theimmunosorbent was equilibrated in 0.1 M sodium phosphate buffer at pH7.0 and packed into a 5.0× 30.0 cm chromatographic column equipped withsliding flow adaptors to a height of 6 cm. To prevent nonspecificadsorption of protein, the immunosorbent was treated by the sequentialaddition of 20 ml. of normal serum, 0.1 M phosphate buffer at pH 7.0,3.0 M ammonium thiocyanate in 0.1 M phosphate buffer at pH 7.0 andfinally reequilibrated with 0.1 M phosphate buffer, pH 7.0.

Immobilized Reactive Dye Chromatography

Immobilized reactive dye adsorbent chromatography was carried oututilizing as the immobilized reactive dye Sepharose-blue dextran.Sepharose-blue dextran was prepared by covalently linking Blue Dextran2000 (Pharmacia) to Sepharose 4B. The product was packed into a 5 cm×13cm column. The sample from the affinity chromatography procedure waspumped through the column at the rate of 120 ml./hr. to remove most ofthe albumin present. Fractions of 10 ml. were collected. The gel bed waswashed free of unabsorbed protein. A solution of 6 M urea in the columnbuffer, i.e., 0.0 s M TRIS, i.e., 2-amino-2-(hydroxy-methyl)-1,3-propanediol, and 0.05 M sodium chloride was utilized to desorb thealbumin from the column thereby regenerating it. The AFP-containingfractions of the unadsorbed eluate were pooled and concentrated byultrafiltration. To insure total removal of the albumin, the unadsorbedfraction was rechromatographed on the column. The column was againregenerated as described above.

In this step and in subsequent steps, the purification of AFP wasmonitored by analytical disc electrophoresis and by immunologictechniques, e.g., immunoelectrophoresis, double immunodiffusion and RIA.

Gel Filtration Chromatography

The unadsorbed fraction from the Sepharose-blue dextran column wasconcentrated and dialyzed to a volume of approximately 20 ml. bydiscontinuous diafiltration against 0.05 M monobasic sodiumphosphate--0.15 M sodium chloride buffer, pH 5.0. The dialyzed samplewas chromatographed to remove high molecular weight contaminants bypassage through a 5.0 cm×85.0 cm column packed with Sephadex G-200(Pharmacia), previously equilibrated with the phosphate saline bufferdescribed above, at an upward flow rate of 60 ml./hr. The eluate fromthe column was collected in 10 ml. fractions. The fractions (150 in all)were monitored for protein content and radioactivity. Fractions 100through 120, which were found to have the highest AFP content, werecollected and the rest discarded.

Ion Exchange Cellulose Chromatography

A mixed bed cationic exchanger (carboxymethyl cellulose)/anionicexchanger (diethylaminoethyl cellulose) medium was equilibrated with 0.1M ammonium acetate at pH 6.25 and gravity packed into a 2.5 cm×13.0 cmcolumn. The pooled fractions from the previous procedure wereequilibrated with the ammonium acetate buffer and applied to the columnat a flow rate of 60 ml./hr. After addition of the pooled fractions wascomplete, 150 ml. of pure buffer were applied to the column. Thereaftera linear salt gradient was established by gradually increasing theproportion of a buffer of 0.5 M sodium chloride in 0.1 M ammoniumacetate at pH 6.25 in the stream of pure buffer. As the proportion ofsalt in the buffer flow increases, proteins are removed from the columnin accordance with their electrostatic binding capacity. Fractions of 3ml. were collected and monitored for protein content and radioactivityas in the previous procedure. The AFP-containing fractions werecollected and pooled.

Preparative Acrylamide Electrophoresis

The pooled fractions from the preceding step were dialyzed againstdeionized water and lyophilized. The resulting powder was dissolved in2.0 ml. of a mixture of 1 part of a 40% by weight aqueous solution ofsucrose containing a trace of bromophenol blue and 3 parts ofelectrophoresis buffer which contains in each liter 6.0 g. TRIS, i.e.,2-amino-2-(hydroxymethyl)-1,3-propanediol, and 28.8 g. glycine, pH 8.3.

A Buchler Polyslab (Buchler Instruments, Inc.) which comprises averticle 170 mm×3.0 mm slab of 7% polyacrylamide resolving gel (smallpore) on top of which is a 10 mm×3.0 mm polyacrylamide concentrating gel(large pore) was equilibrated with the electrophoresis buffer. Thesolution containing the sample was loaded onto the Polyslab and putunder a constant current of 40 mA for 24 hours. During electrophoresis,the proteins in the sample initially collected as a fine line in theconcentrating gel, then gradually dispersed and separated in theresolving gel due to differences in molecular weight, size and charge.

After electrophoresis was completed, the resolving gel was sliced into0.5 cm wide slices transverse to a path between the cathode and theanode. The slices were then eluted with 0.05 M borate buffer, pH 8.4.The eluates were monitored for radioactivity and AFP content by doubleimmunodiffusion and RIA techniques. The eluates containing highlypurified AFP were concentrated by ultrafiltration to approximately 3 ml.

EXAMPLE 2

Individual serum samples obtained from monkies having a chemicallyinduced hepatoma were stored at -20° C. The AFP concentration of thesamples was determined to be in the range of 0.6 to 3.2 mg./ml. by RIAprocedure. The samples were thawed and centrifuged at 100,000 r.p.m. for1 hour. Taking the maximum value obtained by RIA as a basis, thesupernatant was diluted to a content of 0.08 mg AFP/ml. with 0.1 Msodium phosphate buffer containing 0.02% by weight sodium azide, pH 7.0.

Ten ml. portions of the diluted monkey serum (containing a total of 0.8mg. AFP-RIA activity) were cycled through an automated recyclingchromatographic system in accordance with the procedure of Example 1.The adsorbed fractions were concentrated and them chromatographedthrough an immobilized reactive dye adsorbent column, i.e., aSepharose-blue dextran adsorbent column in accordance with the procedureof Example 1.

The unadsorbed fraction from the immobilized reactive dye procedure wasdialyzed and lyophilized. The resulting powder was dissolved in 2.0 ml.of the sucrosebromophenol blue-electrophoresis. The eluates wereconcentrated by ultrafiltration to yield 3 ml. containing highlypurified AFP. Specific activity greater than 0.94 was achieved.

I claim:
 1. A process for producing purified alpha₁ -fetoproteincomprising:(a) buffering a sample of a biological source material knownto contain alpha₁ -fetoprotein to a pH of about 7; (b) subjecting saidbuffered sample to affinity chromatography by passage through a columncontaining an antibody specific for alpha₁ -fetoprotein immobilized on asolid support and subseqently eluting the bound alpha₁ -fetoprotein bytreatment of the column with a suitable desorbing agent; (c) removingsubstantially all of the albumin present in the eluate from step (b) bysubjecting it to adsorption chromatography by passage through a columncontaining an immobilized reactive dye adsorbent; (d) dialyzing theunadsorbed alpha₁ -fetoprotein-containing solution from step (c); (e)lyophilizing the dialyzed solution from step (d); (f) dissolving thepowder formed in step (e) in a suitable buffer and subjecting ehresulting solution to polyacrylamide gel electrophoresis.
 2. The processaccording to claim 1 wherein said source material is monkey hepatomaserum.
 3. The process according to claim 1 wherein the alpha₁-fetoprotein content of the sample is monitored by immunoassay aftersaid steps (b), (c) and (f).
 4. The process according to claim 1 whereinsaid desorbing agent is selected from the group consisting of: aqueoussolutions of urea having a urea concentration of from about 6 to about 8molar; aqueous solutions of sodium chloride having a sodiumconcentration of not less than about 0.5 molar; buffers having a pH offrom about 2 to about 3 and buffers containing a chaotropic ion.
 5. Theprocess in accordance with claim 4 wherein said desorbing agent is abuffer containing a chaotropic ion, said buffer being a 0.1 M sodiumphosphate buffer at about pH 7.0 and said chaotropic ion being thethiocyanate ion.
 6. The process according to claim 1 wherein said solidsupport for said alpha₁ -fetoprotein specific antibody is selected fromthe group consisting of agarose gels, porous glass beads andpolyacrylamide beads.
 7. The process according to claim 1 wherein saidbuffer in step (f) comprises an aqueous solution containing about 0.6%by weight 2-amino-2-(hydroxymethyl)-1,3-propanediol and about 3.0%glycine having a pH of about 8.3.
 8. The process in accordance withclaim 1 wherein said polyacrylamide gel electrophoresis comprisespassage of said sample sequentially through a large pore polyacrylamideconcentrating gel and a small pore polyacrylamide resolving gel overabout 24 hours under a constant current of about 40 mA.
 9. The processin accordance with claim 8 wherein the alpha₁ -fetoprotein is recoveredby eluting portions of said resolving gel with 0.05 M borate buffer atpH 8.4 and concentrating the eluates by ultrafiltration.
 10. The processin accordance with claim 1 wherein said reactive dye is a sulfonated,polyaromatic dye containing a 1, 3, 5-triazine nucleus and a singlereactive chlorine.
 11. The process according to claim 10 wherein saidreactive dye has the structure ##STR2##
 12. The process according toclaims 1, 10 or 11 wherein said immobilized reactive dye adsorbentcomprises an agarose gel covalently linked to dextran having boundthereto a dye having the structure ##STR3##
 13. A process for producingpurified alpha₁ -fetoprotein comprising:(a) buffering a sample of abiological source material known to contain alpha₁ -fetoprotein to a pHof about 7; (b) subjecting said buffered sample to affinitychromatography by passage through a column containing an antibodyspecific for alpha₁ -fetoprotein immobilized on a solid support andsubsequently eluting the bound alpha₁ -fetoprotein by treatment of thecolumn with a suitable desorbing agent; (c) removing substantially allof the albumin present in the eluate from step (b) by subjecting it toadsorption chromatography by passage through a column containing animmobilized reactive dye adsorbent; (d) dialyzing the unadsorbed alpha₁-fetoprotein-containing soution from step (c); (e) removing highmolecular weight protein contaminants from the dialyzed olution fromstep (d) by subjecting it to gel filtration chromatography utilizing asthe filtering material a hydrophilic, water-insoluble, cross-linkeddextran polymer gel; (f) treating the eluate containing alpha₁-fetoprotein from step (e) to remove protein contaminants by subjectingit to ion exchange chromatography utilizing as the filtering material amixture of a cationic exchange resin and an anionic exchange resin; (g)dialyzing the eluate containing alpha₁ -fetoprotein from step (f); (h)lyophilizing the dialyzed solution from step (g); (i) dissolving thepowder formed in step (h) in a suitable buffer and subjecting theresulting solution to polyacrylamide gel electrophoresis.
 14. Theprocess according to claim 10 wherein said source material is cord bloodor cord serum.
 15. The process according to claim 10 wherein the alpha₁-fetoprotein content of the samples is monitored by immunoassay aftersaid steps (b), (c), (e), (f) and (i).
 16. The process according toclaim 10 wherein said buffer in step (d) is a 0.05 M sodium phosphatebuffer containing 0.15 M sodium chloride and 0.02% by weight sodiumazide.
 17. The process according to claim 10 wherein said desorbingagent is selected from the group consisting of: aqueous solutions ofurea having a urea concentration of from about 6 to about 8 molar;aqueous solutions of sodium chloride having a sodium concentration ofnot less than about 0.5 molar; buffers having a pH of from about 2 toabout 3 and buffers containing a chaotropic ion.
 18. The process inaccordance with claim 14 wherein said desorbing agent is a buffercontaining a chaotropic ion, said buffer being a 0.1 M sodium phosphatebuffer at about pH 7.0 and said chaotropic ion being the thiocyanateion.
 19. The process according to claim 10 wherein said solid supportfor said alpha₁ -fetoprotein specific antibody is selected from thegroup consisting of agarose gels, porous glass beads and polyacrylamidebeads.
 20. The process according to claim 10 wherein in step (d) thesolution is dialyzed against the buffer utilized in step (a).
 21. Theprocess according to claim 10 wherein said cross-linked dextran polymergel utilized in step (e) has a molecular exclusion of about 200,000, awater regain of 20±2.0 g. water/g. dry gel and a particle size of 40-120microns.
 22. The process according to claim 10 wherein said cationicexchange resin utilized in step (f) is carboxymethyl cellulose and saidanionic exchange resin is diethylaminoethyl cellulose.
 23. The processin accordance with claim 19 wherein the alpha₁ -fetoprotein solutionobtained from step (e) are dialyzed against a 0.1 M ammonium acetatebuffer, pH about 6.25 being applied to the column in step (f) and, aftersaid column has been washed with said buffer, the alpha₁ -fetoprotein iseluted therefrom by treating the column with an increasing gradient ofsodium chloride in said buffer.
 24. The process according to claim 10wherein said buffer in step (i) comprises an aqueous solution containingabout 0.6% by weight 2-amino-2-(hydroxymethyl)-1,3-propanediol and about3.0% glycine having a pH of about 8.3.
 25. The process in accordancewith claim 10 wherein said polyacrylamide electrophoresis comprisespassage of said sample sequentially through a large pore polyacrylamideconcentrating gel and a small pore polyacrylamide resolving gel over 24hours under a constant current of about 40 mA.
 26. The process inaccordance with claim 22 wherein the alpha₁ -fetoprotein is recovered byeluting portions of said resolving gel with 0.05 M borate buffer at pH8.4 and concentrating the eluates by ultrafiltration.
 27. The process inaccordance with claim 13 wherein said reactive dye is a sulfonated,polyaromatic dye containing a 1, 3, 5-triazine nucleus and a singlereactive chlorine.
 28. The process according to claim 27 wherein saidreactive dye has the structure ##STR4##
 29. The process according toclaims 13, 27 or 28 wherein said immobilized reactive dye adsorbentcomprises an agarose gel covalently linked to dextran having boundthereto a dye having the structure ##STR5##
 30. Radioiodinated alpha₁-fetoprotein suitable for use in a human radioimmuno-assay comprisingalpha₁ -fetoprotein purified from monkey source material in accordancewith the method of claim 1 and radiolabeled with iodine
 125. 31.Radioiodinated alpha₁ -fetoprotein inaccordance with claim 24 whereinsaid monkey source material is monkey hepatoma serum.
 32. In aradioimmunoassay for the alpha₁ -fetoprotein in an unknowncomprising:(a) forming a complex of the alpha₁ -fetoprotein in theunknown with an antiserum; (b) adding thereto radiolabeled alpha₁-fetoprotein; (c) removing the resulting complex from solution; and (d)measuring the amount of radiolabeled alpha₁ -fetoprotein taken upagainst a standard the improvement which comprises utilizing as saidradiolabeled alpha₁ -fetoprotein purified according to the process ofclaim
 10. 33. In a radioimmunoassay for the alpha₁ -fetoprotein in anunknown comprising:(a) forming a complex of the alpha₁ -fetoprotein inthe unknown with antiserum; (b) adding thereto radiolabeled alpha₁-fetoprotein; (c) removing the resulting complex from solution; and (d)measuring the amount of radiolabeled alpha₁ -fetoprotein taken upagainst a standard the improvement which comprises utilizing as saidradiolabeled alpha₁ -fetoprotein purified from monkey source materialaccording to the method of claim
 1. 34. The improved radioimmunoassayaccording to claim 27 wherein said monkey source material is monkeyhepatoma serum.